Material and method for removing immunoglobulins from whole blood

ABSTRACT

An immunoadsorbent material comprising inactivated, protein-A bearing Staphlococcus aureus immobilized in a polymeric matrix is used to remove IgG immunoglobulins and immune complexes from whole blood.

This is division of application Ser. No. 209,872 filed Nov. 24, 1980 andnow U.S. Pat. No. 4,409,330.

BACKGROUND OF THE INVENTION

Protein A-bearing strains of Staphlococcus aureus contain a cell wallpolypeptide that binds to the Fc region of many mammalian IgG subclassesand has a high affinity for immune complexes. Recently it has beenreported that use of such bacteria in an extracorporeal blood processingtechnique results in tumor regression in canine mammary adenocarcinoma(Terman et al, The Journal of Immunology, 124, 795-805 (1980)) andreversal of feline leukemia when the technique is used in combinationwith low dose irradiation (Jones et al., Cancer, 46, 675-684 (1980)).

The blood processing technique involves withdrawing a portion of thetotal blood volume of the tumor bearing host, separating the blood intoplasma and formed elements by centrifugation, running the separatedplasma through a biological filter containing heat-killed andformalin-fixed protein A-bearing Staphlococcus aureus, and thereafterrecombining the treated plasma and formed elements into whole bloodwhich is returned to the host subject.

Although the mechanism of the tumor regressions reported remainsunclear, it has been established that serum IgG levels in treatedsubjects decline immediately after perfusion of the plasma over thebacteria and then rebound above preperfusion levels. Regardless of themechanism(s) involved, the ex vivo removal of serum IgG and immunecomplexes appears to be a key factor in the observed clinicalimprovement in the subjects studied.

The disadvantage of the prior art blood processing technique is that itrequires separation of plasma from formed elements prior to perfusion ofthe plasma. Such a separation step is not only time consuming but alsoincreases the possibility of contamination which could be critical tothe treated subject.

It has now been discovered that IgG and immune comlexes can be removedfrom whole blood thereby eliminating the need to separate the blood intoformed elements and plasma prior to removal of the substances from theplasma.

SUMMARY OF THE INVENTION

The present invention relates to an immunoadsorbent material and use ofsame for removing immunoglobulins and immune complexes from whole blood.

Accordingly, a first aspect of the present invention relates to animmunoadsorbent material comprising inactivated, protein A-bearingStaphlococcus aureus bacteria immobilized in a polymeric matrix.

A second aspect of invention relates to a method of treating whole bloodto remove IgG immunoglobulins and immune complexes which methodcomprises contacting the blood with the immunoadsorbent material of thepresent invention.

The removal of IgG immunoglobulins and immune complexes from whole bloodhas wide ranging use applications. Because the immunoadsorbent binds theimmunoglobulins reversibly, the bound material removed from the bloodcan be subsequently released from the immunoadsorbent and recovered inpure form by a simple change in pH or ion strength. The presentinvention can also be utilized to separate one or more immunoglobulinsof the IgG class from other immunoglobulins present in the blood.

Still another use application of the present invention involves ex vivoremoval of IgG and immune complexes from the blood of mammals afflictedwith neoplastic tumors. As discussed earlier, such treatment appears toretard the growth of neoplasms in subjects afflicted with same.

Accordingly, a further aspect of the present invention relates to amethod of retarding the growth of neoplasms in subject mammals afflictedwith same, which method comprises contacting said subject's blood in aextra-corporeal circulating system with an immnoadsorbent materialcomprising inactivated, protein A-bearing Staphlococcus aureus bacteriaimmobilized in a polymeric matrix and thereafter transfusing saidtreated blood into the host subject.

The term "immune complex" as used herein refers to an antigen/antibodycomplex wherein the antibody is an IgG immunoglobulin.

DETAILED DESCRIPTION

The bacterium employed in the preparation of the immunoadsorbentmaterial of the present invention is a protein A-bearing Staphlococcusaureus. While most strains of Staphlococcus aureus synthesize protein A,there is marked variation in amount in individual strains. One of thebest producers is Staphlococcus aureus Cowan I (ATCC-12598), which isthe preferred strain for use in the present invention.

The Staphlococcus aureus organisms, grown in growth-medium at 37° C. arewashed with phosphate buffered saline and then formalin-stabilized andheat killed. After fixing, the bacterial cells are stored at 4° C. inphosphate buffered saline containing 0.05% azide.

The immunoadsorbent material of the present invention is prepared byadding a 0.1-20% solution of a suitable polymer in aqueous media to anaqueous suspension of formalin-stabilized, heat-killed protein A-bearingStaphlococcus aureus. If necessary, the resulting aqueous suspension maybe heated to effect complete solution of the polymer. Polymers that maybe employed in the present invention include, for example, agarose,chitosan, polyacrylamide, polyacrylamide derivatives and the like.

The above prepared aqueous suspension is then added to a waterimmiscible solvent with agitation. If the aqueous suspension has beenheated to effect solution of the polymer, the solvent should also beheated prior to addition of the suspension to the solvent. Suitablesolvents that may be used include, for example, n-hexane, xylene and thelike. Agitation of the resulting dispersion results in aqueous dropletsof bacteria and polymer dispersed in water immiscible solvent.Dispersion in certain instances may be facilitated by the presence of adetergent.

The aqueous droplets can be hardened with resultant bead formation bycooling the dispersion to room temperature or by the addition of agelling agent or cross-linking agent. Gelling agents that may beemployed include, for example, acetic anhydride, formaldehyde and thelike. Cross-linking agents that may be employed include glutaraldehyde,divinyl sulfone, epichlorohydrin and the like.

The immunoadsorbent material of the present invention can be used toremove IgG immunoglobulins and immune complexes from whole blood bysimply contacting the blood with the material.

Such contact is effected by passing the blood over a column packed withbeads of the immunoadsorbent material or by mixing the blood with anamount of such beads in a suitable container. The duration of thecontact is not bound to critical limits although its value should ofcourse be sufficient to allow all IgG and immune complexes to beadsorbed onto the beads; this duration may normally vary between 1 to 10minutes.

IgG immunoglobulins and immune complexes bound to the immunoadsorbentcan be readily released and recovered by eluting a column of the boundmaterial with 1M acetic acid/PBS buffer, pH 3.; 0.1M glycine-HCL buffer,pH 3, may also be used for desorption of bound material.

When the immunoadsorbent material of the present invention is used toremove IgG and immune complexes from the blood of a tumor bearing hostmammal, removal is effected utilizing an extracorporeal immunoadsorptionsystem. In such a system, a volume of blood from the tumor bearing hostis withdrawn, pumped through a column of immunoadsorbent beads andthereafter collected and transfused back into the host subject byintravenous route.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The following specific description is given to enable those skilled inthe art to more clearly understand and practice the present invention.It should not be considered as a limitation upon the scope of theinvention but merely as being illustrative and representative thereof.

EXAMPLE 1

Staphlococcus aureus Cowan I bacteria (ATCC 12598) are grown inTodd-Hewitt broth, pH 7.9 (Scott Laboratories, Fiskeville, R.I.) whichhas been sterilized at 121° C. for 15 minutes. Two hundred fifty mlEhrlenmeyer flasks containing 50 ml sterilized broth per flask areinnoculated from primary broth cultures grown at 37° C. withoutagitation (the primary cultures have been innoculated from Todd-Hewittagar slant cultures). The resultant 50 ml cultures are incubated at 37°C. with agitation (100 rpm) on a shaker having a circular orbit.Bacterial growth is monitored using a Bausch and Lomb Spectronic-20spectrophotometer, i.e., samples are withdrawn at 15 minute intervalsand the optical density (O.D.) determined at a wavelength of 550 nm.Growth is allowed to proceed until no further increase in O.D. isobserved in two consecutive samples. Average cell yields areapproximately 10 g (wet weight)/liter.

The cells are collected and washed twice by centrifucation at 9000 rpmfor 15 minutes in phosphate-buffered saline (PBS, 150 mM NaCl, 40 mMphosphate), pH 7.2, containing 0.05% (w/v) sodium azide. Afterresuspension to approximately a 10% (w/v) concentration in PBS-azide,the cells are stirred at 23° C. for 1.5 hours in the presence of 1.5%formalin, washed, and again resuspended to the same concentration inbuffer without formalin. The cells are then added to a large Ehrlenmeyerflask to a depth of less than 1.5 cm and killed by rapid swirling in an80° C. water bath for 5 minutes, followed by rapid cooling in anice-water bath. After 2 more washes in PBS-azide, the cell suspension isadjusted to a 10% (v/v) concentration. If not used immediately, the cellsuspension is stored at 4° C.

EXAMPLE II

The cell suspension prepared according to EXAMPLE I is sedimented andresuspended in water several times in order to remove most of thephosphate buffered saline. After the final sedimentation, sufficientwater is decanted so that the settled cells occupy approximately 60% ofthe total volume. Thereafter, 25 ml of the cell suspension is quicklyheated to approximately 80° C. and 25 ml of a hot 3% solution of agarosein water is added to the cell suspension with stirring. The resultingaqueous suspension is immediately added to one liter of warm (i.e., 35°C.) n-hexane containing 100 ml of Witcamide 511 (Witco Chemical Corp.).The hexane mixture is shaken vigorously for approximately 5 minutes oruntil the formed beads adequately harden. The hexane solution is thendecanted from the settled beads which are washed twice with hexane andthen three times with methanol. The beads are suspended in water and,with the aid of water rinses, passed through sieves of successivelydecreasing mesh size to afford gel-like beads having the followingdiameters: 600-800μ (2 ml); 300-600μ (10 ml); 150-300μ (15 ml); and lessthan 150μ (10 ml).

EXAMPLE III

A sample of heparinized human whole blood assayed for its total contentof IgG and IgG complexes is found to contain 10.2 mg per ml of plasma or5.6 mg per ml of blood. Ten ml of the whole blood sample is passedthrough a small column holding a 1.0 ml bed volume of immunoadsorbentbeads (diameter=150-300μ) resting on a loosely packed plug of glasswool.

Previous testing of a similar column with a sample of standardized humanserum established that 2.6 mg of IgG was bound. It was thus estimatedthat the column treated with the 10 ml human blood sample would bindless than 5% of the total IgG fraction in the blood sample.

After the 10 ml blood sample is passed through the column, the column iswashed three times with 2 ml portions of PBS buffer, pH 7.2. The secondand third washes are negative for IgG. Thereafter the column is washedwith 1 ml of 1M acetic acid/PBS buffer, pH 3.0, followed by 4 ml of PBSbuffer at pH 7.2.

The eluted material is quantitated with commercial immunodiffusionplates (Calbiofchem Behring Corp., La Jolla, CA.) after neutralizationto pH 7. The eluted material is found to contain 2.2 mg IgG.

In a parallel experiment, an attempt was made to pass a 10 ml sample ofthe whole blood through a similar sized column holding a 1 ml bed volumeof the commercial immunoadsorbent, Protein A-Sepharose® CL-4B (PharmaciaFine Chemicals, Piscataway, N.J.). The initial flow of blood wasextremely slow and stopped after approximately 2 ml passed through.

What is claimed is:
 1. A method of treating whole blood to remove IgGimmunoglobulins and immune complexes, which method comprises contactingsaid blood with an immunoadsorbent material comprising inactivated,protein A-bearing Staphlococcus aureus bacteria immobilized in apolymeric matrix said material being in the form of gel like beadshaving an average diameter ranging from 100 to 1000 microns.
 2. A methodaccording to claim 1 wherein said polymeric matrix is selected from thegroup consisting of agarose, chitosan, polyacrylamide and polyacrylamidederivatives.
 3. A method according to claim 2 wherein said polymericmatrix is agarose.
 4. A method according to claim 1 wherein said bloodis contacted using column chromatography that employs theimmunoadsorbent as the solid phase.
 5. A method of retarding the growthof neoplasts in subject mammals suffering from same, which methodcomprises:a. contacting the blood of said subjects in an extra-corporealcirculating system with an immunoadsorbent material comprisinginactivated, protein A-bearing Staphlococcus aureus bacteria immobilizedin a polymeric matrix said material being in the form of gel like beadshaving an average diameter ranging from 100 to 1000 microns and b.transfusing said subjects contacted blood back into the host subject. 6.A method according to claim 5 wherein said polymeric matrix is selectedfrom the group consisting of agarose, chitosan, polyacrylamide andpolyacrylamide derivatives.
 7. A method according to claim 6 whereinsaid polymeric matrix is agarose.